The cuticular methyL-branched alkanes of tsetse flies of the subgenera Glossina (sensu stricto, formerly morsitans) and Nemorhina (formerly palpalis) were identified and quantified by capillary gas–liquid chromatography (GC) and gas chromatography–mass spectrometry (GC-MS). Males of Glossina (Nemorhina) are differentiated from G. (Glossina) by dominant 27-, 28-, and/or 29-carbon backbone trimethylalkanes with the methyl positions at 3,7,11-, 4,8,12-, and 3,7,11-, respectively. All females contain major quantities of long-chain internally branched di- and/or trimethylalkanes that were previously implicated as mediators of sexual behavior in males. Taxa within these two subgroups that are closely related and/or conspecific, based upon conventional morphological and ecological criteria, exhibit similar GC patterns and similar internally branched di- and trimethylalkane isomers in females. Examination of these potentially stimulatory methylalkanes may provide reasons for the reproductive isolation of closely related species from each other.